Omomyc interacts with Myc and Max in cells and also affects Myc stability and protein levels. (A) Immunoprecipitation (IP) of Myc, Max, and biotinylated Omomyc in HCT116 cells. HCT116 cells were treated with 10 μM Omomyc, 2.5 μM Omomyc with ProteoJuice, or no Omomyc at all and then lysed after 24 h. Lysates were then incubated with antibodies against Myc and Max, control IgG, and streptavidin; pulled down with magnetic beads; run on an SDS-PAGE gel; and then blotted with antibodies to Myc, Max, or biotin. (B) Effect of treatment with 10 μg/ml cycloheximide in the presence and absence of 10 μM Omomyc over time. HCT116 cells were treated with 10 μg/ml cycloheximide in the presence or absence of 10 μM Omomyc for up to 4 h. Cells were lysed, subjected to SDS-PAGE, and then Western blotted with antibodies to Myc, Max, and Gapdh. (C) Effect of cycloheximide or the proteasome inhibitor MG132 on Myc, Max, and Omomyc interactions. HCT116 cells were treated with 0 or 10 μM Omomyc in the absence or presence of 20 μg/ml cycloheximide or 10 μM MG132 for 4 h; lysed; incubated with antibodies (ab) to Myc and Max, control IgG, and streptavidin; and then pulled down with magnetic beads. The material was run on an SDS-PAGE gel and Western blotted with antibodies to Myc, Max, and biotin. (D) Treatment of Ramos cell lysates with Omomyc induces thermal destabilization of Myc. Shown are aggregation temperature (Tagg) curves and corresponding Western blot images for Myc in Ramos cell lysates after incubation at 4°C with Omomyc (20 μM) or PBS for 2 h. All data were normalized to the Myc band intensity for the lowest temperature, 39°C. The Tagg shifts were analyzed using the Boltzmann sigmoidal equation and are 50.3°C for the buffer-treated lysate and 43.9°C for the Omomyc-treated lysate. (E) Omomyc decreases the level of Myc in the cell. Ramos cells were treated with increasing concentrations of Omomyc for 24 h, lysed, run on an SDS-PAGE gel, and Western blotted with antibodies to Myc and Gapdh. (F) Time course of Myc reduction by 10 μM Omomyc. Ramos cells were treated with Omomyc, and samples were collected at 0, 2, 4, and 24 h. The samples were lysed, subjected to SDS-PAGE, and Western blotted for Myc and Gapdh. (G) Myc reduction is due to proteosomal degradation of Myc. Ramos cells were treated with or without 10 μM Omomyc in the presence or absence of 100 nM MG132 for 24 h, lysed, subjected to SDS-PAGE, and Western blotted for Myc and Gapdh.